Targeting genes responsible for vectorial capacity is a novel approach to controlling infectious diseases. To develop a better understanding of genetic determinants of vectorial capacity and with support from vector biologists and members of the malaria community, NHGRI and NIAID have funded the sequencing of the genomes and transcriptomes of 16 Anopheles species (http://www.broadinstitute.org/annotation/genome/anopheles). With genome sequences becoming available, researchers now have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, success of these comparative genomic analyses will be limited, and inferences about evolution of the vectorial capacity traits will be less informative if researchers deal with numerous sequencing scaffolds rather than with chromosome-based genome assemblies. The major goal of this R21 project is to develop chromosome-based reference genome assemblies for three major malaria vectors: An. arabiensis, An. stephensi, and An. albimanus. Draft genome assemblies are available for these species (https://olive.broadinstitute.org/comparisons/anopheles.1). Our project is timely because it will create crucial genomic tools for the newly sequenced Anopheles species. The project is innovative because it will use the automated multicolor fluorescent in situ hybridizatio (AM-FISH) and automated microscopic analysis. We will, for the first time, perform phylogenetic analysis of anopheline mosquitoes by genome-wide gene order analysis and gain important insights into patterns and mechanisms of chromosomal rearrangements. The availability of state-of-the-art equipment and the expertise of the PI in cytogenetics and comparative genomic analysis will ensure successful achievement of the project's goal. The proposal has three specific aims. Specific Aim 1. Physically map sequencing scaffolds to chromosomes of An. arabiensis, An. stephensi, and An. albimanus. We will place and orient scaffolds to polytene chromosomes by AM-FISH, thus, creating chromosome-based reference genome assemblies for the species belonging to the series Pyretophorus (subgenus Cellia), series Neocellia (subgenus Cellia), and series Albimanus (subgenus Nyssorhynchus), respectively. Specific Aim 2. Reconstruct genome-scale rearrangement phylogeny of genus Anopheles. We will identify breakpoints of all fixed inversions in An. arabiensis and will refine the chromosomal phylogeny in the An. gambiae complex using An. stephensi and An. albimanus as outgroup species. Specific Aim 3. Determine the pattern and mechanisms of chromosome evolution in genus Anopheles. We will test the hypotheses that (i) the X chromosome has the highest rate of inversion fixation despite the paucity of inversion polymorphisms, and (ii) inversions have chromosome-specific and species-specific mechanisms of origin and rates of fixation.